Next to applications in fermentations, Lactobacillus plantarum is recognized as a food spoilage organism, and its dispersal from biofilms in food processing environments might be implicated in contamination or recontamination of food products. This study provides new insights into biofilm development by L. plantarum WCFS1 through comparative analysis of wild type and mutants affected in cell surface composition, including mutants deficient in the production of Sortase A involved in the covalent attachment of 27 predicted surface proteins to the cell wall peptidoglycan (ΔsrtA) and mutants deficient in the production of capsular polysaccharides (CPS1–4, Δcps1–4). Surface adhesion and biofilm formation studies revealed none of the imposed cell surface modifications to affect the initial attachment of cells to polystyrene while biofilm formation based on Crystal Violet (CV) staining was severely reduced in the ΔsrtA mutant and significantly increased in mutants lacking the cps1 cluster, compared to the wild-type strain. Fluorescence microscopy analysis of biofilm samples pointed to a higher presence of extracellular DNA (eDNA) in cps1 mutants and this corresponded with increased autolysis activity. Subsequent studies using Δacm2 and ΔlytA derivatives affected in lytic behaviour revealed reduced biofilm formation measured by CV staining, confirming the relevance of lysis for the build-up of the biofilm matrix with eDNA.